Uneven cellular expression of recombinant α2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition

AbstractTwo populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (α2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number

Recombinant human α2-adrenoceptor subtypes: comparison of [3H]rauwolscine, [3H]atipamezole and [3H]RX821002 as radioligands

AbstractKinetic, saturation and competition binding assays were employed to optimize and validate radioligand binding methods for characterization of recombinant human α2-adrenoceptor subtypes and for screening of new subtype-selective ligands. Stable transfected lines of Shionogi 115 mouse mammary tumour cells (5115) and three structurally different antagonist radioligands, [3H]rauwolscine, [3H]atipamezole and [3H]RX821002, were used. Specificity of α2-adrenergic binding was defined with 100 μM (−)-adrenaline. Steady-state was reached with all three radioligands within 15–30 min at 25°C, and the binding was rapidly reversible. The receptor affinities (α2-C10) were highest in glycylglycine, almost equally high in K+-phosphate, and lowest in Tris buffer for all three [3H]-ligands. This was mainly caused by different association rates. [3H]RX821002 was bound with high affinity and similar kinetic properties to all three αa2adrenoceptor subtypes in K+-phosphate buffer, and had the highest proportion of specific binding (96–98%). [3H]RX821002 and K+-phosphate buffer were subsequently used in competition assays. The rank order of affinity of compounds selective for α2-adrenoceptor subtypes was α2-C1O > α2-C4 > α2-C2 for oxymetazoline, α2-C4 > α2-C2 > α2-C10 for prazosin and α2-C2 > α2-C4 > α2-C10 for chlorpromazine. The drug affinities (Ki values) determined in this system were in close agreement with earlier results with [3H]rauwolscine in Tris buffer (r = 0.94). Agonist competition for [3H]RX821002 binding was biphasic in K+-phosphate buffer supplemented with 10 MM MgCl2, indicating functional coupling of receptors to G-proteins. Accordingly high-affinity binding of the agonists (−)-noradrenaline and UK14,304 was eliminated by 10 μM Gpp(NH)p in the assays. Our results confirm that [3H]RX821002 is a suitable radioligand for the characterization of all three human α2-adrenoceptor subtypes and for the determination of the subtype-selectivity of new α2-adrenoceptor agonists and antagonists

The insertion/deletion variation in the α(2B)-adrenoceptor does not seem to modify the risk for acute myocardial infarction, but may modify the risk for hypertension in sib-pairs from families with type 2 diabetes

BACKGROUND: An insertion/deletion polymorphism in the α(2B)-adrenoceptor (AR) has been associated with the risk for acute myocardial infarction (AMI) and sudden cardiac death. In this study we tested whether this polymorphism is associated with the risk for AMI among members of families with type 2 diabetes. METHODS: 154 subjects with a history of AMI were matched for age and sex with one of their siblings who did not have a history of AMI. The prevalence of the genotypes of the α(2B)-AR insertion/deletion polymorphism was compared between the siblings using McNemar's test. We also explored the data to see whether this genetic variation affects the risk for hypertension by using logistic regression models in the two subpopulations of subjects, with and without a history of AMI. RESULTS: Among all study subjects, 73 (24%) carried the α(2B)-AR deletion/deletion genotype, 103 (33%) carried the insertion/insertion genotype, and 132 (43%) were heterozygous. The distribution of genotypes of the α(2B)-AR insertion/deletion variation in the group of subjects with a history of AMI and their phenotype-discordant siblings did not statistically significantly differ from that expected by random distribution (p = 0.52): the deletion/deletion genotype was carried by 34 subjects with AMI (22%), and by 39 subjects without AMI (25%). Neither did we observe any significant difference in deletion allele frequencies of the α(2B)-AR insertion/deletion polymorphism between patients with a history of AMI (0.44) and their sib-pair controls (0.46, p = 0.65). In an exploratory analysis, the α(2B)-AR deletion/deletion genotype was associated with increased odds for hypertension compared with subjects carrying any of the other genotypes. CONCLUSIONS: The deletion/deletion genotype of the α(2B)-AR does not emerge in this study as a risk factor for AMI among members of families with type 2 diabetes; however, it might be involved in the development of hypertension

Treating gambling disorder with as needed administration of intranasal naloxone : a pilot study to evaluate acceptability, feasibility and outcomes

Correction:Background and aim There is growing interest in the use of medication-assisted treatments for gambling disorder (GD). Opioid receptor antagonists are hypothesised to blunt the craving associated with gambling. This study was designed to assess the feasibility of using an intranasal naloxone spray to treat GD. Design An 8-week, open-label, uncontrolled pilot study. Setting A single study site in the capital region of Finland. Subjects Twenty problem gamblers (nine men) were randomised into two groups. Group A (n=10) took one dose into one nostril (2mg naloxone), as needed, with a maximum of 4 doses/day (max. 8mg/day). Group B (n=10) took one dose into each nostril (4mg naloxone) as needed, with a maximum of 4 doses/day (max. 16mg/day). Intervention Naloxone hydrochloride nasal spray. Measures Acceptability and feasibility of the intervention were assessed. Use of study medication, adverse events, gambling frequency and gambling expenditure were recorded in a mobile diary. Problem gambling: South Oaks Gambling Screen (SOGS), depressive symptoms: Beck Depression Inventory (BDI) and alcohol use: Alcohol Use Disorders Identification Test were recorded. Results Study completion rate was 90%. Acceptability and feasibility scores were high. Group B used intranasal naloxone more frequently than group A, and consequently used more naloxone. No serious adverse events were reported. The postintervention SOGS scores were lower (median=4 (IQR=3.75) versus preintervention scores (median=12 (IQR=4.75)). Depressive symptoms were reduced during the trial (preintervention BDI median=9, IQR=9vs postintervention BDI median=6, IQR=6). Conclusions The acceptability and feasibility of using intranasal naloxone were high, and no serious adverse events were reported. Preliminary results suggest mixed results in terms of gambling behaviour (ie, reduced frequency but not expenditure) and decreased depressive symptoms. Trial registration number EudraCT2016-001828-56Peer reviewe

Acute stress effects of impulsive noise during mental work

Impulsive sound has been found to annoy people more than steady-state sound or many other types of sound presented at the same sound level. This study examined the physiological, performance, and subjective effects of impulsive sound on working humans. Exposure to impulsive sound (65 dB LAeq) was compared with quiet sound (35 dB LAeq) and steady-state sound (65 dB LAeq). This parallel-group study, where each participant was exposed to one sound condition, had altogether 59 participants. Physiological stress was measured with stress hormone concentrations in plasma (cortisol and noradrenaline), heart rate variability (HRV), and blood pressure. Psychological stress was measured with subjective noise annoyance, workload, and fatigue. Performance was measured in tasks requiring constant concentration (visual and auditory serial recall and N-back). Compared to quiet sound, impulsive sound caused more annoyance, workload, and lack of energy, raised cortisol concentrations, reduced systolic blood pressure, and decreased accuracy in the 3-back task. Compared with steady-state sound, impulsive sound was experienced as more annoying and causing a higher workload and more lack of energy. Impulsive sound caused physiological and psychological stress and decreased performance compared to quiet sound. Part of this load was due to the increased sound level, which was evident as a physiological stress reaction. Still, there was also an extra stress effect related to the impulsiveness of the sound, reflected as a psychological experience. Special care should be paid to impulsive sound, especially in environments where people are performing mental work.</p

Population Modelling of Dexmedetomidine Pharmacokinetics and Haemodynamic Effects After Intravenous and Subcutaneous Administration

Background and Objective: Dexmedetomidine is a potent agonist of α2-adrenoceptors causing dose-dependent sedation in humans. Intravenous dexmedetomidine is commonly used perioperatively, but an extravascular route of administration would be favoured in palliative care. Subcutaneous infusions provide desired therapeutic plasma concentrations with fewer unwanted effects as compared with intravenous dosing. We aimed to develop semi-mechanistic population models for predicting pharmacokinetic and pharmacodynamic profiles of dexmedetomidine after intravenous and subcutaneous dosing.Methods: Non-linear mixed-effects modelling was performed using previously collected concentration and haemodynamic effects data from ten (eight in the intravenous phase) healthy human subjects, aged 19–27 years, receiving 1 µg/kg of intravenous or subcutaneous dexmedetomidine during a 10-min infusion.Results: The absorption of dexmedetomidine from the subcutaneous injection site, and distribution to local subcutaneous fat tissue was modelled using a semi-physiological approach consisting of a depot and fat compartment, while a two-compartment mammillary model explained further disposition. Dexmedetomidine-induced reductions in plasma norepinephrine concentrations were accurately described by an indirect response model. For blood pressure models, the net effect was specified as hyper- and hypotensive effects of dexmedetomidine due to vasoconstriction on peripheral arteries and sympatholysis mediated via the central nervous system, respectively. A heart rate model combined the dexmedetomidine-induced sympatholytic effect, and input from the central nervous system, predicted from arterial blood pressure levels. Internal evaluation confirmed the predictive performance of the final models, as well as the accuracy of the parameter estimates with narrow confidence intervals.Conclusions: Our final model precisely describes dexmedetomidine pharmacokinetics and accurately predicts dexmedetomidine-induced sympatholysis and other pharmacodynamic effects. After subcutaneous dosing, dexmedetomidine is taken up into subcutaneous fat tissue, but our simulations indicate that accumulation of dexmedetomidine in this compartment is insignificant.</p

Effects of intramuscular vatinoxan (MK-467), co-administered with medetomidine and butorphanol, on cardiopulmonary and anaesthetic effects of intravenous ketamine in dogs

Objective To investigate the impact of intramuscular (IM) co-administration of the peripheral alpha(2)-adrenoceptor agonist vatinoxan (MK-467) with medetomidine and butorphanol prior to intravenous (IV) ketamine on the cardiopulmonary and anaesthetic effects in dogs, followed by atipamezole reversal. Study design Randomized, masked crossover study. Animals A total of eight purpose-bred Beagle dogs aged 3 years. Methods Each dog was instrumented and administered two treatments 2 weeks apart: medetomidine (20 mu g kg(-1)) and butorphanol (100 mu g kg(-1)) premedication with vatinoxan (500 mu g kg(-1); treatment MVB) or without vatinoxan (treatment MB) IM 20 minutes before IV ketamine (4 mg kg(-1)). Atipamezole (100 mu g kg(-1)) was administered IM 60 minutes after ketamine. Heart rate (HR), mean arterial (MAP) and central venous (CVP) pressures and cardiac output (CO) were measured; cardiac (CI) and systemic vascular resistance (SVRI) indices were calculated before and 10 minutes after MVB or MB, and 10, 25, 40, 55, 70 and 100 minutes after ketamine. Data were analysed with repeated measures analysis of covariance models. A p-value Results At most time points, HR and CI were significantly higher, and SVRI and CVP significantly lower with MVB than with MB. With both treatments, SVRI and MAP decreased after ketamine, whereas HR and CI increased. MAP was significantly lower with MVB than with MB; mild hypotension (57-59 mmHg) was recorded in two dogs with MVB prior to atipamezole administration. Sedation, induction, intubation and recovery scores were not different between treatments, but intolerance to the endotracheal tube was observed earlier with MVB. Conclusions and clinical relevance Haemodynamic performance was improved by vatinoxan co-administration with medetomidine-butorphanol, before and after ketamine administration. However, vatinoxan was associated with mild hypotension after ketamine with the dose used in this study. Vatinoxan shortened the duration of anaesthesia.Peer reviewe

Nitrogen Balance after the Administration of a Prolonged-Release Protein Substitute for Phenylketonuria as a Single Dose in Healthy Volunteers

Nitrogen balance is the difference between nitrogen excreted as urea and nitrogen ingested, mainly in proteins. Increased circulating concentrations of amino acids (AA) in the bloodstream are usually associated with proportional increases in the production and excretion of urea. Previously, we reported results from a randomized, controlled, single-dose, crossover trial in healthy adult volunteers (n = 30) (Trial Registration: ISRCTN11016729), in which a Test product (prolonged-release AA mixture formulated with Physiomimic Technology (TM) (PT (TM))) significantly slowed down the release and reduced the peak plasma concentrations of essential AAs compared with a free AA mixture (Reference product) while maintaining essential AA bioavailability. Here, we report an assessment of the nitrogen balance from the same study. The amount of nitrogen contained in plasma AAs, levels of blood urea nitrogen (BUN) (p < 0.0001) and changes in BUN (p < 0.0001) were smaller after the Test product compared with the Reference product. These findings suggest that the production of urea in proportion to systemic AA availability was significantly smaller after the administration of the Test product compared with the Reference product and that the test product conferred the increased utilization of AAs for protein synthesis and reduced their oxidation and conversion to urea. In the clinical setting, it is possible that the effects of PT (TM) observed on the disposition of free AAs in this study may translate to health benefits in terms of physiological body composition and growth if used for the treatment of subjects with phenylketonuria (PKU). Further investigation in patients with PKU is warranted

Gene expression profiles and signaling mechanisms in α2B-adrenoceptor-evoked proliferation of vascular smooth muscle cells

Background: alpha(2)-adrenoceptors are important regulators of vascular tone and blood pressure. Regulation of cell proliferation is a less well investigated consequence of alpha(2)-adrenoceptor activation. We have previously shown that alpha B-2-adrenoceptor activation stimulates proliferation of vascular smooth muscle cells (VSMCs). This may be important for blood vessel development and plasticity and for the pathology and therapeutics of cardiovascular disorders. The underlying cellular mechanisms have remained mostly unknown. This study explored pathways of regulation of gene expression and intracellular signaling related to alpha B-2-adrenoceptor-evoked VSMC proliferation.Results: The cellular mechanisms and signaling pathways of alpha B-2-adrenoceptor-evoked proliferation of VSMCs are complex and include redundancy. Functional enrichment analysis and pathway analysis identified differentially expressed genes associated with alpha B-2-adrenoceptor-regulated VSMC proliferation. They included the upregulated genes Egr1, F3, Ptgs2 and Serpine1 and the downregulated genes Cx3cl1, Cav1, Rhoa, Nppb and Prrx1. The most highly upregulated gene, Lypd8, represents a novel finding in the VSMC context. Inhibitor library screening and kinase activity profiling were applied to identify kinases in the involved signaling pathways. Putative upstream kinases identified by two different screens included PKC, Raf-1, Src, the MAP kinases p38 and JNK and the receptor tyrosine kinases EGFR and HGF/HGFR. As a novel finding, the Src family kinase Lyn was also identified as a putative upstream kinase.Conclusions: alpha B-2-adrenoceptors may mediate their pro-proliferative effects in VSMCs by promoting the activity of bFGF and PDGF and the growth factor receptors EGFR, HGFR and VEGFR-1/2. The Src family kinase Lyn was also identified as a putative upstream kinase. Lyn is known to be expressed in VSMCs and has been identified as an important regulator of GPCR trafficking and GPCR effects on cell proliferation. Identified Ser/Thr kinases included several PKC isoforms and the beta-adrenoceptor kinases 1 and 2. Cross-talk between the signaling mechanisms involved in alpha(2) B-adrenoceptor-evoked VSMC proliferation thus appears to involve PKC activation, subsequent changes in gene expression, transactivation of EGFR, and modulation of kinase activities and growth factormediated signaling. While many of the identified individual signals were relatively small in terms of effect size, many of them were validated by combining pathway analysis and our integrated screening approach